The present invention relates to the field of selective protection of active groups on chemical compounds. In particular, the present invention provides unique reagents and methods for selectively protecting active groups in chemical synthesis.
It is well-known that proteins are significantly involved in many of the bodily states in multicellular organisms, including most disease states. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutic methods have generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is to employ oligonucleotides, especially oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence, to modulate, enhance or otherwise affect translation.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to functioning as both indirect and direct regulators of proteins, oligonucleotides have also found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides, via Watson-Crick and/or Hoogsteen base pairs, to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides as primers in polymerase chain reactions (PCR) has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied.
For example, PCR technology is used in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides, both natural and synthetic, are employed as primers in PCR technology.
Laboratory uses of oligonucleotides are described generally in laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA (see Book 2 of Molecular Cloning, A Laboratory Manual, ibid.) and DNA-Protein Interactions and The Polymerase Chain Reaction (see Vol. 2 of Current Protocols In Molecular Biology, ibid).
Oligonucleotides can be custom-synthesized for a desired use. Thus a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, (Tm)); to assist in identification of the oligonucleotide or an oligonucleotide-target complex; to increase cell penetration; to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides; to provide a mode of disruption (terminating event) once sequence-specifically bound to a target; and to improve the pharmacokinetic properties of the oligonucleotides.
Thus, it is of increasing value to prepare oligonucleotides and other phosphorus-linked oligomers for use in basic research or for diagnostic or therapeutic applications. Consequently, and in view of the considerable expense and time required for synthesis of specific oligonucleotides, there has been a longstanding effort to develop successful methodologies for the preparation of specific oligonucleotides with increased efficiency and product purity.
Synthesis of oligonucleotides can be accomplished using both solution phase and solid phase methods. Oligonucleotide synthesis via solution phase in turn can be accomplished using various coupling mechanisms. However, solution phase chemistry requires purification after each internucleotide coupling, which is labor-intensive and time consuming.
The current method of choice for the preparation of naturally occurring oligonucleotides, as well as modified oligonucleotides such as phosphorothioate and phosphorodithioate oligonucleotides, is via solid-phase synthesis, wherein an oligonucleotide is prepared on a polymer support (a solid support) such as controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373); or POROS, a polystyrene resin available from Perceptive Biosystems. Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide chain. Typically, a first nucleoside (having protecting groups on any exocyclic amine functionalities present) is attached to an appropriate glass bead support and activated phosphite compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide. The nucleotide phosphoramidites are reacted with the growing oligonucleotide using xe2x80x9cfluidized bedxe2x80x9d technology to mix the reagents. The known silica supports suitable for anchoring the oligonucleotide are very fragile and thus cannot be exposed to aggressive mixing. Additional methods for solid-phase synthesis may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Kxc3x6ster U.S. Pat. Nos. 4,725,677 and Re. 34,069.
Phosphoramidites typically have been prepared by one of three routes. In the first, a suitably protected nucleobase is reacted with a protected bis-dialkylamino phosphite compound in the presence of 1H-tetrazole or a tetrazole salt. See Nielsen, J. et al., Nucleic Acids Res. 1986, 14, 7391; Nielsen, J. et al., J. Chem. Res.(S) 1986, 26; Hamamoto, S. et al., Chem. Lett. 1986, 1401; and Nielsen, J. et al., Nucleic Acids Res. 1987, 15, 3626. This method is disadvantageous because, inter alia, tetrazole is a health hazard, and poses disposal problems due to its explosive nature.
A second method for the preparation of phosphoramidites involves reacting the 3xe2x80x2-hydroxyl of a nucleoside with a protected dialklyamino chloro phosphitylting reagent. See Hering, G. et al., Nucleosides Nucleotides 1985, 4, 169; and Ugi, I. et al., J. Chem. Soc. Chem. Commun. 1997, 877. This method also is disadvantageous because of the explosive nature of the phosphitylating reagent.
A third method for the synthesis of phosphoramidites involves reacting the 3xe2x80x2-hydroxyl of a nucleoside with a dialklyamino dichloro compound, followed by displacement of chlorine with addition of a protecting group. Tanaka, T. et al., Tetrahedron Lett. 1986, 27, 199. Phosphorodiamidites also can be prepared, for example, by the condensation of a bis(dialkylamino) chlorophosphine with a 5xe2x80x2-protected nucleoside according to the procedure of Grandas et al., Tetrahedron Letters 1989 30 (5) 543-546.
The chemical structures of the five 2xe2x80x2-dexoynucleosides and four 2xe2x80x2-O-alkyl nucleosides that are considered essential raw materials for the preparation of antisense drugs are shown below. 
The lengthy and inefficient harvesting process of nucleosides from fish has led to several attempts to achieve their production via a combination of chemical and enzymatic approaches. Although such strategies are used for the manufacturing of pyrimidine-containing 2xe2x80x2-deoxynucleosides (e.g. T, dC and 5-Me dC), they are not efficient for production of purine-containing deoxynucleosides (such as dA and dG). This problem has prompted the development of alternative processes, in which dA and dG could be obtained via chemical deoxygenation of the corresponding ribonucleotides A and G, respectively, that are easily manufactured in ton scales from yeast RNA. Nonetheless, the latter approach requires a C2xe2x80x2 deoxygenation step that is most efficient using a 3xe2x80x2-, 5xe2x80x2-protected nucleoside precursor.
There has been increasing attention directed toward a class of 2xe2x80x2-O-substituted ribonucleic acids, such as 2xe2x80x2-O-methyl and 2xe2x80x2-O-methoxyethyl-substituted ribonucleic acids. Incorporation of 2xe2x80x2-O-alkylated ribonucleotides in antisense oligonucleotides increases both resistance of the oligonucleotides to nucleases and their thermal stability with complementary mRNA. See E. L. Ruff et al., Journal of Organic Chemistry, 1996, 61, 1547-1550;, H. Cramer et al., Helvetica Chimica Acta, 1996, 79, 2114-2136. As a result, considerable effort has been directed toward investigation of 2xe2x80x2-O-alkylated nucleoside-containing oligonucleotides. As the 2xe2x80x2-O-alkylated nucleosides represent more than 60% of the cost of raw materials in the synthesis of the respective oligonucleotides, the reduction in cost of 2xe2x80x2-O-alkylated nucleosides is of great importance for the further investigation and exploitation of these useful nucleosides.
Preparation of 2xe2x80x2-O-alkylated nucleosides requires alkylation of the 2xe2x80x2-OH of the corresponding nucleosides. The 2xe2x80x2-O-alkylation process is problematic and inefficient, especially for large-scale manufacturing, and particularly where the 2xe2x80x2-O-alkylated nucleoside is a purine nucleoside, such as 2xe2x80x2-methyladenosine (2xe2x80x2-Me A), 2xe2x80x2-methoxyethyladenosine (2xe2x80x2-MOE A), 2xe2x80x2-methyl guanosine (2xe2x80x2-Me G), or 2xe2x80x2-methoxyethoxyguanosine (2xe2x80x2-MOE G). P. Martin, Helvetica Chimica Acta 1995, 78, 486-504; U. Legorburu et al., Tetrahedron 1999, 55, 5635-5640.
The problems associated with the production of 2xe2x80x2-deoxygenated and 2xe2x80x2-O-alkylated purine nucleosides have been addressed by selective protection of the 3xe2x80x2- and 5xe2x80x2-hydroxyl groups. Heretofore, the most convenient protecting group for such protection has been tetraiosopropyldichlorodisiloxane (1, TIPDSCl2), also known as Markiewicz reagent. 
Despite the advantages of TIPDSCl2, its high cost and its fragility during basic treatment make it incompatible with the conditions necessary for alkylation at the C2xe2x80x2-oxygen of ribonucleosides. Due to these limitations, TIPDSCl2 is not generally used during the manufacture of 2xe2x80x2-O-alkylated ribonucleosides.
There is thus a need for a protecting group that is capable of protecting the 5xe2x80x2- and 3xe2x80x2-hydroxyl groups of a nucleoside under the basic conditions required for 2xe2x80x2-O-alkylation of ribonucleosides.
There is also a need for an economical protecting group that is capable of protecting the 5xe2x80x2- and 3xe2x80x2-hydroxy groups of a nucleoside during 2xe2x80x2-deoxygenation process in the synthesis of deoxynucleotides.
There is also a need for a protecting group that is capable of protecting multiple reactive groups during synthesis.
There is also a need for a protecting group that is capable of protecting reactive groups under a variety of conditions.
There is also a need for an improved process of selectively alkylating the 2xe2x80x2-hydroxyl of a nucleoside.
There is also a need for an improved process of deoxygenating a nucleoside at the 2xe2x80x2-position.
There is also a need for an improved process of selectively protecting one or more reactive groups on a chemical compound during organic synthesis.
There is also a need for processes of synthesizing protective groups capable of protecting two reactive groups on a nucleoside.
There is also a need for 3xe2x80x2,5xe2x80x2-diprotected nucleoside intermediates useful in the production of 2xe2x80x2-deoxy and/or 2xe2x80x2-modified nucleosides.
There is further a need for an improved electrophile for preparing 2xe2x80x2-O-methyl nucleosides.
The foregoing and other needs are met by embodiments of the present invention, which provide a process of using a compound of the formula I to protect one or more active groups on a precursor, said compound of formula I being: 
wherein A is a moiety other than O; each of L1 and L2 is independently a leaving group; and each of R1-R4 is independently a substituent, or two of R1-R4 taken together on the same or different Si form a silicon-containing ring; said process comprising a step for reacting the compound of formula I with said precursor to form a protected intermediate, said precursor having at least one reactive group.
The foregoing and other needs are further met by embodiments of the present invention, which provide a compound comprising a residue of a precursor having at least one reactive group coupled to a residue of a compound of formula (I), above.
The foregoing and other needs are further met by embodiments of the present invention, which provide synthetic methods of using a precursor to make a product, said precursor having at least a first and a second reactive group, the methods comprising reacting said precursor with a compound of formula (I) for a time and under conditions sufficient to protect said first reactive group, whereby a protected intermediate is formed, subjecting the protected intermediate to conditions and/or contacting said intermediate with at least one reagent for acting on the second reactive group to produce a protected product, and deprotecting the protected product to form the product.
The foregoing and other needs are further met by embodiments of the present invention, which provide synthetic methods of using a precursor to make a product, said precursor having at least a first and a second reactive group, the methods comprising reacting said precursor with a compound of formula (I) for a time and under conditions sufficient to protect said first reactive group, whereby a protected intermediate is formed, subjecting the protected intermediate to conditions and/or contacting said intermediate with at least one reagent for acting on the second reactive group to produce a protected product, and deprotecting the protected product to form the product, wherein the conditions and/or reagents for acting on the second reactive group remove the reactive group.
The foregoing and other needs are further met by embodiments of the present invention, which provide synthetic methods of using a precursor to make a product, said precursor having at least a first and a second reactive group, the methods comprising reacting said precursor with a compound of formula (I) for a time and under conditions sufficient to protect said first reactive group, whereby a protected intermediate is formed, subjecting the protected intermediate to conditions and/or contacting said intermediate with at least one reagent for acting on the second reactive group to produce a protected product, and deprotecting the protected product to form the product, wherein the conditions and/or reagents for acting on the second reactive group alkylate the reactive group.
The foregoing and other needs are met by embodiments according to the present invention, which provide compounds of the formula (II): 
wherein A is a moiety other than O and R1-R4 are as defined in formula I, Y is H, OH or a 2xe2x80x2-substituent in the arabino- or ribo-configuration and Bx is a nucleobase.
The foregoing and other needs are met by embodiments according to the present invention, which provide compounds of formula III: 
wherein each of R1-R4 is defined in formula I, A is a moiety other than O as defined herein and Bx is a nucleobase.
The foregoing and other needs are met by embodiments of the present invention, which provide compounds of formula IV: 
wherein each of R1-R4 is defined in formula I, A is a moiety other than O as defined herein and Bx is a nucleobase.
The foregoing and other needs are met by embodiments of the present invention, which provide compounds of formula V: 
wherein each of R1-R4 is defined in formula I, A is a moiety other than O as defined herein, Y is a 2xe2x80x2-deoxy-2xe2x80x2-substituent in the ribo- or arabino-configuration and Bx is a nucleobase.
The foregoing and other needs are met by embodiments according to the present invention, which provide compounds of the formula (VI): 
wherein A is a moiety other than O and R1-R4 are as defined in formula I above, Bx is a nucleobase, G3 is S, O or NR5, R5 is H or alkyl, and Z is a 2xe2x80x2-O-substituent.
The foregoing and other needs are met by embodiments according to the present invention, which provide compounds of formula VII: 
wherein each of R1-R4 is alkyl, Z is a 2xe2x80x2-O-substituent, A is a moiety other than O as defined herein and Bx is a nucleobase.
The foregoing and other needs are met by embodiments of the present invention, which provide compounds of formula VIII: 
wherein each of R1-R4 is lower alkyl, Z is lower alkyl, substituted lower alkyl, or lower acyl or substituted lower acyl, A is a moiety other than O as defined herein and Bx is a nucleobase.
The foregoing and other needs are met by embodiments of the present invention, which provide compounds of formula IX: 
wherein each of R1-R4 is independently lower alkyl, Z is methyl or methoxyethyl, A is CH2, and Bx is a nucleobase.
The foregoing and other needs are met by embodiments of the present invention, which provide methods of preparing a compound of the formula: 
wherein Bx is a nucleobase,
said method comprising reacting a ribonucleoside of formula: 
wherein Bx is a nucleobase,
with a compound of formulas I to form a compound of formula IV, performing one or more steps for removing the 2xe2x80x2-OH to form a compound of one of the formulae III, and removing the bis-silyl protecting group to form the compound of formula IX.
The foregoing and other needs are further met by a process of making a 2xe2x80x2-alkylated ribonucleoside of the formula X: 
wherein Bx is a nucleobase and Z is a 2xe2x80x2-O-substituent, such as alkyl, substituted alkyl, acyl or protected acyl,
said process comprising the steps of:
reacting a ribonucleoside, with a bis-silyl compound of formula I, to form a protected intermediate of one of formulae IV,
reacting the protected intermediate with a compound of the formula X-Z, wherein X is a leaving group and Z is a 2xe2x80x2-O-substituent to form a protected product of formula VII or VIII, above, and then removing the bis-silyl protecting group to provide the compounds of formula X.
The foregoing and other needs are further met by embodiments of the present invention, which provide methods of manufacturing substituted nucleosides of formula X, said methods comprising reacting a compound of the formula: 
wherein A is a linking moiety, such as O or a moiety other than O, and R1-R4 and L1 and L2 are all defined in formula I,
with a nucleoside to form a 5xe2x80x2,3xe2x80x2-protected nucleoside of the formula: 
wherein A, R1-R4 and Bx are as defined above,
reacting the 5xe2x80x2,3xe2x80x2-protected nucleoside with a compound of the formula:
X-Z,
wherein X is a leaving group and Z is a 2xe2x80x2-O-substituent, such as alkyl, substituted alkyl, acyl or substituted acyl,
in the presences of a mild hindered base or a salt thereof, to form a protected intermediate of the formula: 
and removing the 5xe2x80x2,3xe2x80x2-protective group to produce the product of formula X.
The foregoing and other objects are further met by embodiments of the present invention, which provide a method of making a 2xe2x80x2-O-methyl nucleoside, the process comprising reacting a ribonucleoside with a silylating agent of formula I to form a protected intermediate of formula IV, (wherein Z is CH3), reacting the protected intermediate with an electrophile in the presence of a mild hindered base, and removing the protecting group to produce a 2xe2x80x2-O-methyl nucleoside. In some preferred embodiments of the present invention, the mild hindered base is hexamethyldisilazane (HMDS) or a salt thereof. In some especially preferred embodiments, the mild hindered base is sodium HMDS (NaHMDS). In some preferred embodiments, the electrophile is a methyl halide. In especially preferred embodiments of the invention, the electrophile is methyl chloride (CH3Cl).
The foregoing and other objects are further met by embodiments of the present invention which provide methods of manufacturing compounds according to formula (I), as further described herein.